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Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4.

Yang J, Qin N, Zhang H, Yang R, Xiang B, Wei Q - Sci Rep (2016)

Bottom Line: These results indicate that the uptake of exogenous CnB did not occur through LPS and that CnB was not a chaperone of LPS.Thus, we conclude that TLR4 receptor complexes were required for the recognition and internalization of exogenous CnB.These properties of CnB support its potential for development as an anti-cancer drug.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, P. R. of China.

ABSTRACT
Our previous research showed that recombinant calcineurin B (rhCnB) stimulates cytokine secretion by immune cells, probably through TLR4. Exogenous CnB can be incorporated into many different tumour cells in vitro, but the mode of uptake and receptors required remain unknown. Here, we report that exogenous CnB is taken up by cells in a time- and concentration-dependent manner via clathrin-dependent receptor-mediated internalization. Our findings further confirm that uptake is mediated by the TLR4/MD2 complex together with the co-receptor CD14. The MST results revealed a high affinity between CnB and the TLR4 receptor complex. No binding was detected between CnB and LPS. CnB inhibited the uptake of LPS, and LPS also inhibited the uptake of CnB. These results indicate that the uptake of exogenous CnB did not occur through LPS and that CnB was not a chaperone of LPS. Thus, we conclude that TLR4 receptor complexes were required for the recognition and internalization of exogenous CnB. CnB could be a potential endogenous ligand of TLR4 and function as an agonist of TLR4. These properties of CnB support its potential for development as an anti-cancer drug.

No MeSH data available.


Related in: MedlinePlus

The uptake of exogenous CnB occurred via TLR4 receptor-mediated internalization.(a) Co-localization of rhodamine-labelled CnB with clathrin-GFP (upper panel) or CnB-GFP with rhodamine-labelled transferrin (lower panel) in SK-HEP-1 cells. The clathrin-GFP transfected cells were co-incubated with 5 μM CnB-rhodamine, or SK-HEP-1 cells were co-incubated with 5 μM CnB-GFP mixed with 5 μM rhodamine-labelled transferrin for 30 min, and visualized using a confocal laser scanning microscope (×63, scale bar 10 μm). (b) Free CnB inhibits the uptake of the fluorescently labelled CnB. The cells were co-incubated with excess CnB and DyLight 488-labeled CnB or labelled CnB alone for 30 min and visualized using an inverted fluorescence microscope (upper panel, scale bar 50 μm, 20×). The fluorescence intensity was quantified using a microplate reader (lower panel). (c) Positive correlation between CnB uptake and TLR4 expression. 5 × 105 cells from different cell lines were co-incubated with 5 μM CnB for 10 min, subjected to Trizol treatment and RNA extraction. Extracted mRNA was used for qPCR analysis of TLR4. The qPCR results were analyzed and compared with CnB-GFP uptake (lower panel). 5 × 106 cells from different cell lines were co-incubated with 5 μM CnB for 10 min and lysed with RIPA buffer, the samples were used for detecting the protein level of TLR4 by western blot analysis (upper panel). (d) Co-localization of exogenous CnB-GFP and TLR4-cherry. The TLR4-cherry- or cherry-transfected Hek293 cells were co-incubated with 5 μM CnB-GFP for 30 min, and visualized using a confocal laser scanning microscope (63×, scale bar 20 μm). (e,f) Effect of TLR4 knock down on CnB uptake. The influence of TLR4 knock down on CnB uptake was analysed by western blot analysis (e) or FI (f), (scale bar 50 μm, 20×). (g) TAK242 inhibited CnB uptake. The SK-HEP-1 cells were pre-incubated with 10 μM or 5 μM TAK242 or vehicle for 3 h, followed by co-incubation with CnB-GFP for 30 min. The co-incubated cells were washed, acid-stripped and quantified by a microplate reader. Bars represent mean ± s.e.m. from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 (t-test, two-tailed).
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f2: The uptake of exogenous CnB occurred via TLR4 receptor-mediated internalization.(a) Co-localization of rhodamine-labelled CnB with clathrin-GFP (upper panel) or CnB-GFP with rhodamine-labelled transferrin (lower panel) in SK-HEP-1 cells. The clathrin-GFP transfected cells were co-incubated with 5 μM CnB-rhodamine, or SK-HEP-1 cells were co-incubated with 5 μM CnB-GFP mixed with 5 μM rhodamine-labelled transferrin for 30 min, and visualized using a confocal laser scanning microscope (×63, scale bar 10 μm). (b) Free CnB inhibits the uptake of the fluorescently labelled CnB. The cells were co-incubated with excess CnB and DyLight 488-labeled CnB or labelled CnB alone for 30 min and visualized using an inverted fluorescence microscope (upper panel, scale bar 50 μm, 20×). The fluorescence intensity was quantified using a microplate reader (lower panel). (c) Positive correlation between CnB uptake and TLR4 expression. 5 × 105 cells from different cell lines were co-incubated with 5 μM CnB for 10 min, subjected to Trizol treatment and RNA extraction. Extracted mRNA was used for qPCR analysis of TLR4. The qPCR results were analyzed and compared with CnB-GFP uptake (lower panel). 5 × 106 cells from different cell lines were co-incubated with 5 μM CnB for 10 min and lysed with RIPA buffer, the samples were used for detecting the protein level of TLR4 by western blot analysis (upper panel). (d) Co-localization of exogenous CnB-GFP and TLR4-cherry. The TLR4-cherry- or cherry-transfected Hek293 cells were co-incubated with 5 μM CnB-GFP for 30 min, and visualized using a confocal laser scanning microscope (63×, scale bar 20 μm). (e,f) Effect of TLR4 knock down on CnB uptake. The influence of TLR4 knock down on CnB uptake was analysed by western blot analysis (e) or FI (f), (scale bar 50 μm, 20×). (g) TAK242 inhibited CnB uptake. The SK-HEP-1 cells were pre-incubated with 10 μM or 5 μM TAK242 or vehicle for 3 h, followed by co-incubation with CnB-GFP for 30 min. The co-incubated cells were washed, acid-stripped and quantified by a microplate reader. Bars represent mean ± s.e.m. from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 (t-test, two-tailed).

Mentions: Clathrin-mediated endocytosis, which was historically referred to as “receptor-mediated endocytosis”, mediates the internalization of signalling and nutrient receptors, with the best-characterized mechanism being the protein trafficking of ion channels28. To examine whether clathrin mediates CnB uptake, we incubated clathrin-GFP transfected SK-HEP-1 cells with rhodamine-labelled CnB. As shown in the upper panel of Fig. 2a, rhodamine-labelled CnB co-localized with clathrin-GFP; we also used rhodamine-labelled transferrin to trace the clathrin-mediated pathway29. CnB-GFP overlapped with rhodamine-labelled transferrin (lower panel of Fig. 2a). Subsequently, the application of excess CnB to inhibit the uptake of DyLight 488-labeled CnB resulted in significant inhibition of the uptake of CnB-GFP (Fig. 2b), which revealed that CnB uptake required the participation of the receptor. Based on these findings, we conclude that the transport of CnB occurred via clathrin-dependent receptor-mediated internalization.


Cellular uptake of exogenous calcineurin B is dependent on TLR4/MD2/CD14 complexes, and CnB is an endogenous ligand of TLR4.

Yang J, Qin N, Zhang H, Yang R, Xiang B, Wei Q - Sci Rep (2016)

The uptake of exogenous CnB occurred via TLR4 receptor-mediated internalization.(a) Co-localization of rhodamine-labelled CnB with clathrin-GFP (upper panel) or CnB-GFP with rhodamine-labelled transferrin (lower panel) in SK-HEP-1 cells. The clathrin-GFP transfected cells were co-incubated with 5 μM CnB-rhodamine, or SK-HEP-1 cells were co-incubated with 5 μM CnB-GFP mixed with 5 μM rhodamine-labelled transferrin for 30 min, and visualized using a confocal laser scanning microscope (×63, scale bar 10 μm). (b) Free CnB inhibits the uptake of the fluorescently labelled CnB. The cells were co-incubated with excess CnB and DyLight 488-labeled CnB or labelled CnB alone for 30 min and visualized using an inverted fluorescence microscope (upper panel, scale bar 50 μm, 20×). The fluorescence intensity was quantified using a microplate reader (lower panel). (c) Positive correlation between CnB uptake and TLR4 expression. 5 × 105 cells from different cell lines were co-incubated with 5 μM CnB for 10 min, subjected to Trizol treatment and RNA extraction. Extracted mRNA was used for qPCR analysis of TLR4. The qPCR results were analyzed and compared with CnB-GFP uptake (lower panel). 5 × 106 cells from different cell lines were co-incubated with 5 μM CnB for 10 min and lysed with RIPA buffer, the samples were used for detecting the protein level of TLR4 by western blot analysis (upper panel). (d) Co-localization of exogenous CnB-GFP and TLR4-cherry. The TLR4-cherry- or cherry-transfected Hek293 cells were co-incubated with 5 μM CnB-GFP for 30 min, and visualized using a confocal laser scanning microscope (63×, scale bar 20 μm). (e,f) Effect of TLR4 knock down on CnB uptake. The influence of TLR4 knock down on CnB uptake was analysed by western blot analysis (e) or FI (f), (scale bar 50 μm, 20×). (g) TAK242 inhibited CnB uptake. The SK-HEP-1 cells were pre-incubated with 10 μM or 5 μM TAK242 or vehicle for 3 h, followed by co-incubation with CnB-GFP for 30 min. The co-incubated cells were washed, acid-stripped and quantified by a microplate reader. Bars represent mean ± s.e.m. from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 (t-test, two-tailed).
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f2: The uptake of exogenous CnB occurred via TLR4 receptor-mediated internalization.(a) Co-localization of rhodamine-labelled CnB with clathrin-GFP (upper panel) or CnB-GFP with rhodamine-labelled transferrin (lower panel) in SK-HEP-1 cells. The clathrin-GFP transfected cells were co-incubated with 5 μM CnB-rhodamine, or SK-HEP-1 cells were co-incubated with 5 μM CnB-GFP mixed with 5 μM rhodamine-labelled transferrin for 30 min, and visualized using a confocal laser scanning microscope (×63, scale bar 10 μm). (b) Free CnB inhibits the uptake of the fluorescently labelled CnB. The cells were co-incubated with excess CnB and DyLight 488-labeled CnB or labelled CnB alone for 30 min and visualized using an inverted fluorescence microscope (upper panel, scale bar 50 μm, 20×). The fluorescence intensity was quantified using a microplate reader (lower panel). (c) Positive correlation between CnB uptake and TLR4 expression. 5 × 105 cells from different cell lines were co-incubated with 5 μM CnB for 10 min, subjected to Trizol treatment and RNA extraction. Extracted mRNA was used for qPCR analysis of TLR4. The qPCR results were analyzed and compared with CnB-GFP uptake (lower panel). 5 × 106 cells from different cell lines were co-incubated with 5 μM CnB for 10 min and lysed with RIPA buffer, the samples were used for detecting the protein level of TLR4 by western blot analysis (upper panel). (d) Co-localization of exogenous CnB-GFP and TLR4-cherry. The TLR4-cherry- or cherry-transfected Hek293 cells were co-incubated with 5 μM CnB-GFP for 30 min, and visualized using a confocal laser scanning microscope (63×, scale bar 20 μm). (e,f) Effect of TLR4 knock down on CnB uptake. The influence of TLR4 knock down on CnB uptake was analysed by western blot analysis (e) or FI (f), (scale bar 50 μm, 20×). (g) TAK242 inhibited CnB uptake. The SK-HEP-1 cells were pre-incubated with 10 μM or 5 μM TAK242 or vehicle for 3 h, followed by co-incubation with CnB-GFP for 30 min. The co-incubated cells were washed, acid-stripped and quantified by a microplate reader. Bars represent mean ± s.e.m. from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 (t-test, two-tailed).
Mentions: Clathrin-mediated endocytosis, which was historically referred to as “receptor-mediated endocytosis”, mediates the internalization of signalling and nutrient receptors, with the best-characterized mechanism being the protein trafficking of ion channels28. To examine whether clathrin mediates CnB uptake, we incubated clathrin-GFP transfected SK-HEP-1 cells with rhodamine-labelled CnB. As shown in the upper panel of Fig. 2a, rhodamine-labelled CnB co-localized with clathrin-GFP; we also used rhodamine-labelled transferrin to trace the clathrin-mediated pathway29. CnB-GFP overlapped with rhodamine-labelled transferrin (lower panel of Fig. 2a). Subsequently, the application of excess CnB to inhibit the uptake of DyLight 488-labeled CnB resulted in significant inhibition of the uptake of CnB-GFP (Fig. 2b), which revealed that CnB uptake required the participation of the receptor. Based on these findings, we conclude that the transport of CnB occurred via clathrin-dependent receptor-mediated internalization.

Bottom Line: These results indicate that the uptake of exogenous CnB did not occur through LPS and that CnB was not a chaperone of LPS.Thus, we conclude that TLR4 receptor complexes were required for the recognition and internalization of exogenous CnB.These properties of CnB support its potential for development as an anti-cancer drug.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Beijing Normal University, Gene Engineering and Biotechnology Beijing Key Laboratory, Beijing, 100875, P. R. of China.

ABSTRACT
Our previous research showed that recombinant calcineurin B (rhCnB) stimulates cytokine secretion by immune cells, probably through TLR4. Exogenous CnB can be incorporated into many different tumour cells in vitro, but the mode of uptake and receptors required remain unknown. Here, we report that exogenous CnB is taken up by cells in a time- and concentration-dependent manner via clathrin-dependent receptor-mediated internalization. Our findings further confirm that uptake is mediated by the TLR4/MD2 complex together with the co-receptor CD14. The MST results revealed a high affinity between CnB and the TLR4 receptor complex. No binding was detected between CnB and LPS. CnB inhibited the uptake of LPS, and LPS also inhibited the uptake of CnB. These results indicate that the uptake of exogenous CnB did not occur through LPS and that CnB was not a chaperone of LPS. Thus, we conclude that TLR4 receptor complexes were required for the recognition and internalization of exogenous CnB. CnB could be a potential endogenous ligand of TLR4 and function as an agonist of TLR4. These properties of CnB support its potential for development as an anti-cancer drug.

No MeSH data available.


Related in: MedlinePlus